Glutamate dehydrogenase (GDH) is an ancient enzyme found in all living organisms. The underlying chemistry of the reversible oxidative deamination of glutamate to 2-oxoglutarate catalyzed by this enzyme has remained unchanged through the epochs. However, GDH from the animal kingdom is allosterically regulated by a large number compounds while the enzyme from other kingdoms is completely unregulated. Through our individual and collaborative efforts, we have determined the structures of animal GDH complexed with several of these allosteric regulators and have models for how allostery is exacted. We have also shown the importance of GDH regulation by our finding that mutations that affect GTP inhibition are the root cause of the hyperinsulinism/hyperammonemia (HI//HA) syndrome in children. Furthermore, we have shown that GDH is involved in both glucose and leucine stimulated secretion of insulin from normal pancreatic tissue. We propose that this regulation of insulin homeostasis is due to GDH-mediated controlof intracellular signaling molecule, glutamine. It is therefore apparent that understanding the allosteric regulation of GDH is crucial to understanding insulin homeostasis. To that end, this proposal aims to use a combination of techniques to understand where these various regulators bind and how they modulate enzymatic activity. We will also further analyze some of HI/HA mutants to better understand the pathology of this life-threatening disorder. Perhaps most exciting is that we have leveraged our understanding of GDH to discover a family of new, non-toxic compounds that act via GDH and can potentially treat both HI/HA and type II diabetes. Finally, we have shown that the GDH from Ciliates is an evolutionary 'missing link' between animals and the other kingdoms. By further studying this form of GDH we will elucidate animal allostery by better understanding how and why it evolved. Together, these studies will not only answer fundamental questions as to how protein subunits communicate with each other during allostery but will solidify our hypothesis that allosteric regulation of GDH plays a critical role in insulin homeostasis.